1. Field of the Invention
This invention relates to a user-friendly transformable bacteria and production of biological molecules using the transformable bacteria, and specifically this invention relates to a mutant strain of Rhodobacter bacteria capable of uptake and maintenance of heterologous DNA. One application uses invented mutant strains for efficient production of biological molecules.
2. Background of the Invention
The search continues for rapid and efficient means to harness biological systems to produce complex molecules. A myriad of molecule types are targeted for more rapid and efficient production, including but not limited to feedstocks, nutrients, membrane proteins, drop-in fuel moieties, nutraceuticals, specialty chemicals, and precursors of bio-based polymers.
Membrane proteins are particularly relevant targets given their potential as drug development templates. Biological membranes serve as the interface between living matter and the environment. Membranes also separate functional compartments within complex cells. Some organisms produce intracellular membranes which are used to divide cells into functional and structural compartments such as organelles, vacuoles, nuclear envelopes, etc. In Rhodobacter, intracytoplasmic membranes (ICMs) can be induced to form and simultaneously encapsulate heterologously-expressed membrane proteins. The inventors developed and previously disclosed this system in U.S. Pat. No. 6,465,216 which is incorporated in its entirety by reference.
Proteins inserted in membranes carry out essential biological processes including uptake of nutrients, excretion of wastes, signal transduction, response to external stimuli, and energy conversion reactions. Genes encoding membrane proteins represent approximately 30 percent of every genome. Membrane proteins comprise the majority (up to 80 percent by some industrial estimates) of the drug targets that are being pursued currently.
Most drugs are targeted at G-protein coupled receptors (GPCRs), a class of integral membrane proteins for which there is little structural information. The human genome encodes approximately 800 unique GPCRs, and annual world-wide GPCR-targeting drug sales exceed $250 billion. The impact of an efficient membrane protein expression system in the process for discovery of just a single new GPCR-targeting drug could yield substantial revenue.
In addition, markets for bioengineered crop plants have expanded 10 percent annually over the last 10 years, signifying increased global acceptance. Membrane protein expression technology has the potential to enable and speed certification processes for engineered food crops.
Also, procedures for producing next-generation biofuels use enzymatic reactions that are catalyzed by membrane proteins. The impact of an efficient membrane protein expression system on processes to replace petroleum-based fuels will generate large revenue streams.
Membrane proteins are lipid soluble, and therefore require hydrophobic environments for stability and functional integrity. As such, their abundance in native cells or tissues is often very low. Structure determination—that can be used as an important input for rational drug design—for membrane proteins lags far behind that known for soluble proteins by approximately a 100:1. Thus, expression technology leading to the acquisition of structural information is needed for this important target class.
Researchers require milligram quantities of membrane proteins in their native configurations) to adequately study membrane protein structure and function. Every viable strategy for structure/function studies of membrane proteins is dependent upon an efficient means for membrane protein production.
Commercial expression systems are available that are based on the use of prokaryotic hosts E. coli, Bacillus subtilis, and Lactococcus lactis; these systems excel in the production of soluble proteins. More versatile systems that are capable of post-translational modification of expressed proteins (e.g., glycosylation, etc.) use yeast expression hosts. Commercial yeast expression systems employed for soluble proteins include those based on Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces lactis. All of the above organisms are easily cultured on inexpensive media but are not designed for the expression of membrane proteins because they lack inducible membranes that can sequester newly expressed protein.
Unlike the above mentioned prokaryotes, Rhodobacter is an efficient membrane producer. However, Rhodobacter strains are not amenable to chemical transformation or electroporation so as to enable simple generation of expression strains to produce heterologous proteins. The barrier to the introduction of plasmid DNA in host organisms such as Rhodobacter is that they encode enzymes of DNA restriction-modification systems as protection against foreign DNA. The modification enzymes ‘mark’ the cell's own DNA by methylating it to differentiate it from other DNA in the environment. This allows the restriction endonucleases to ‘restrict’ or destroy any double-stranded DNA which enters the cell that is not marked appropriately. All strains of E. coli that are used commonly as cloning and expression hosts have been genetically modified to remove these endogenous restriction-modification enzymes to enable successful transfer and maintenance of foreign DNA in the cells.
The standard method for introduction of foreign DNA comprises incubation of pure DNA with chemically-treated cells to “transform” them to a selectable phenotype that is encoded by a gene on the segment of pure plasmid DNA. In some transformation protocols, the efficiency of this process is increased by application of an electric current, resulting in transformation via “electroporation”.
At present, neither chemical nor electroporetic transformation of Rhodobacter with pure double-stranded plasmid DNA is generally possible. Previous efforts, such as Formari et al J. Bacteriol 152, 89-97 disclosed that chemically competent R. sphaeroides has been transformed with double-stranded plasmid DNA which lack sequences recognized by endogenous restriction enzymes—a limited approach as most plasmids have these sequences. Other researchers, Donohue, T. J. et al Methods in Enzymology, 204, pp. 459-486 (1991), have transformed electro-competent R. sphaeroides. However, as with chemical transformation discussed supra, electrotransformation was successful only when plasmids lacking sites for endogenous restriction enzymes were used.
Again, since most expression plasmids have these sequences and most genes of foreign proteins one desires to express are likely to have these sequences, it has been necessary to introduce DNA by transferring expression plasmids to Rhodobacter strains via conjugal mating with an appropriate E. coli donor strain. In this process, depicted in FIG. 1, cells of the E. coli strain carrying the plasmid DNA are mixed with cells of the Rhodobacter recipient strain. The DNA from the E. coli donor strain enters the Rhodobacter host in a single-stranded form that is not susceptible to endogenous restriction endonucleases. (Thus the foreign DNA is not cleaved and transfer is successful.) Endogenous modification enzymes originating from within the organism methylate, “mark”, or otherwise protect the incoming DNA concomitantly with its replication to form double-stranded DNA.
Unfortunately, the conjugation depicted in FIG. 1 is time-consuming due to the subsequent requirement for performing multiple rounds of colony growth to purify the desired R. sphaeroides transformants from the E. coli donor cells. Repeated re-streaking and growth of single colonies on agar plates can take up to 10 days. Also, these steps cannot be automated or adapted easily to be compatible with higher-throughput cloning and expression procedures. Conjugation techniques are rarely used in typical molecular biology laboratories. As such, this step in the process of using Rhodobacter as an expression vehicle requires special training and is viewed as cumbersome for the general user.
As noted supra, the inventors previously developed a system whereby plasmid vectors are utilized to induce Rhodobacter bacteria to simultaneously produce, and sequester heterologous protein. However, and as noted supra, transfer of the constructed expression plasmid to the host cell via conjugation is a time-consuming and technically-challenging step requiring specialized training of the laboratory worker.
A need exists in the art for an easily used system for the production of biological moieties which generates milligram quantities of target moiety per liter of culture. The system should be capable of being incorporated into a kit, operational within 1-2 days, and does not require high level of skills in the art. The system should be operable with standard vectors, and transportable in frozen state prior to use.